Project Summary Breast cancer metastasis kills over 40,000 women annually and there are very few therapies that efficiently target this process. A hallmark of breast cancer metastasis is an increase in cancer cell migration and invasion, which is driven in part by the small GTPase RhoA. Unfortunately, therapeutic targeting of RhoA has not been successful because it is expressed ubiquitously throughout the body and has no targetable interfaces. The neuroepithelial transforming gene 1 (Net1), is a Rho guanine exchange factor (RhoGEF) that is overexpressed in breast cancer and is required for breast cancer cell motility. Net1 isoforms are uniquely regulated by nuclear sequestration. However, ligand stimulation of quiescent cells allows the Net1A isoform to relocalize to the cytoplasm, where it can activate RhoA. Recent work from our lab has established c-Jun-N-terminal kinase (JNK) and Src signaling are required for ligand stimulated cytosolic localization of Net1A, and that these kinases phosphorylate distinct sites within Net1A to cause this effect. Based on these findings, we hypothesize that Src cooperates with JNK to regulate Net1A cytoplasmic accumulation to promote breast cancer motility and metastasis. In Aim 1, we will determine how Src-dependent phosphorylation of Net1A on Y373 controls its subcellular localization. We will identify the kinase that directly phosphorylates Net1A on this site, and elucidate the mechanism by which this drives Net1A nuclear export. In Aim 2 we will determine how JNK and Src signaling synergize to promote cytoplasmic accumulation of Net1A to stimulate RhoA activation and breast cancer cell motility in vitro. In Aim 3, we will establish the role of JNK- and Src- dependent phosphorylation of Net1A in mammary gland tumorigenesis and metastasis using orthotopic mammary tumor models. We hypothesize these signaling events to be key drivers of Net1A-mediated motility and invasion. Identification of these regulatory mechanisms may provide novel therapeutic approaches to mitigate breast cancer metastasis.